CN111418558B

CN111418558B - Construction method of ablation metabolic urinary calculus animal model

Info

Publication number: CN111418558B
Application number: CN202010379140.XA
Authority: CN
Inventors: 刘耀明; 姚雅
Original assignee: Shanxi University
Current assignee: Shanxi University
Priority date: 2019-09-22
Filing date: 2020-05-07
Publication date: 2021-09-28
Anticipated expiration: 2040-05-07
Also published as: CN111418558A

Abstract

A method for constructing an animal model for ablating metabolic urolithiasis, belonging to the field of biomedicine; it is characterized in that the potassium citrate solution with a concentration of 6.7-12.8 mM and a volume of 5-10 μL is injected into the built-up urolithiasis animal model, and citric acid is injected. After 12 hours of potassium solution, the calcium oxalate crystals formed in the Malpighian canal of the animal model gradually disintegrated until completely disappeared. In order to control the formation process of calcium oxalate crystals, the present invention can regulate the process of gradually disintegrating, ablating and even eliminating the formed calcium oxalate. The animal model construction method of the invention has the advantages of simple operation, short implementation period, readily available materials, reversible and controllable process, and the like, and is suitable for the formation of primary or metabolic urinary calculi and its ablation process, pathological analysis, and drug testing and screening.

Description

Construction method of ablation metabolic urinary calculus animal model

Technical Field

The invention belongs to the field of biomedical research, and particularly relates to a construction method of an ablation metabolic urinary calculus animal model.

Background

The primary or metabolic urinary calculus mainly comprises urinary calculus and kidney calculus, which are mostly generated in people with little water intake, the calculus incidence rate is up to 11%, and in the existing urinary calculus and kidney calculus, calcium oxalate alone or together with other calcium salts forms the main component of urinary calculus. The human being has particularity as a treatment object, deeply understands and researches the formation, prevention and ablation process of the metabolic urinary calculus and needs to rely on a proper animal model to carry out corresponding scientific research. According to the symptom characteristics of calculus in different tissues and organs, different animal models have been constructed using animals such as mice, rabbits, pigs, etc. In order to better understand and understand primary or metabolic urinary calculi, especially the ablation process and physiological mechanism of calculi, it is objectively necessary to construct diversified animal models.

Drosophila became the internationally accepted classical insect model for studying animal diseases and life laws, and provides important reference data for disease occurrence mechanism, treatment scheme and drug screening. The locusta chinensis is a common agricultural pest in a paddy field, and takes stems and leaves of the paddy rice, wherein dozens of hollow tubules with blind ends grow at the junction of intestines and hindgut, and the tubules are dissociated in haemolymph and automatically crawl. The developmental theory and the evolutionary theory believe that the insect Mariothis marxiana and the mammalian kidney are homologous organs, and the genetic background, the physiological structure and the function of the insect Mariothis marxiana are extremely similar, so the Mariothis marxiana is regarded as a detoxifying and urination organ of the insect, small molecule metabolites generated by various tissues in vivo can be absorbed from the haemolymph into the tube to form the prourine, and are combined with midgut metabolites to form the excrement to be discharged out of the body.

In the previous research, the inventor proves that calcium oxalate is formed in the Ma's tube of the Chinese rice locust stressed by copper chloride, and after a certain amount of potassium citrate is injected into the haemolymph of the Chinese rice locust which generates calcium oxalate crystals, the calcium oxalate is gradually ablated. The construction of the insect urinary system stone model based on the physiological process is scientific. The model is applied to develop the ablation process and mechanism of urinary calculus, and provides a test model and a practical carrier for preventing and treating primary or metabolic urinary calculus and screening urinary calculus ablation drugs.

Disclosure of Invention

The invention aims to provide a construction method of an animal model for ablating metabolic urinary calculi, which is a method for ablating calcium oxalate crystals generated in a Ma's tube of a Chinese rice locust by potassium citrate with a certain concentration according to the physiological metabolism rule of the Chinese rice locust on metal ions and application thereof.

A construction method of an animal model for ablating metabolic urinary calculi comprises the following contents:

1. using wild captured adult Chinese rice locust, and adaptively feeding with fresh wheat seedling in a climatic incubator for 2-3 days;

2. the environmental conditions for raising the Chinese rice locust are as follows: the illumination and dark time is 14 h to 10 h, the temperature in illumination is 25-28 +/-2 ℃, the temperature in dark is 21-26 +/-2 ℃, and the humidity is 65-85%;

3. a micro-injection needle penetrates into an internode membrane from the 3 rd and 4 th abdominal joints of the insect abdomen along the direction opposite to the abdomen extension direction and slowly injects a copper chloride solution;

4. the copper chloride solution injected into abdominal cavity of Oxya chinensis (Miq.) Kuntze is prepared from insect normal saline, and is sterilized by steam at 120 deg.C for 20 min;

5. the formula of the insect normal saline comprises: NaCl: 0.138M, Na₂HPO₄：0.0073 M, KH₂PO₄：0.00147 M，KCl：0.0027 M， pH=7.4；

6. Sterilizing with microinjector and insect at 75%, and injecting copper chloride solution into abdominal cavity of Aconitum chinensis (Ornithogalum chinense (L.) Kuntze) at concentration of 6.0-10.5 mM and injection dosage of 4-8 μ L;

7. feeding for 1 day after injecting copper chloride solution, and performing secondary injection of medicinal copper chloride solution by the same method;

8. the parameters of the insect breeding environment after injection are the same as those in the step 2;

9. after the rice is raised for 24 to 48 hours, irregular calcium oxalate crystals are formed in the Ma-tube of the Chinese rice locust, and the crystals show blue fluorescence under ultraviolet and visible light and are cuboid or irregular;

10. the potassium citrate solution is prepared by insect normal saline with the concentration of 12.8 mM, and then is sterilized by steam at 120 ℃ for 20 min;

11. injecting 6.7-12.8 mM potassium citrate solution into Chinese rice locust producing calcium oxalate crystals, wherein the injection volume is 5-10 μ L, and feeding in a climatic incubator;

12. after the Chinese rice locust injected with the potassium citrate solution is raised for 12 hours, dissection and the Marsdenia tenacissima is observed through a microscope, so that calcium oxalate crystals begin to melt and gradually decrease with the time.

Compared with other existing animal models, the invention has the advantages that the adopted Chinese rice locust is a rice field insect, the copper chloride and the potassium citrate are conventional reagents, and the material cost is low; the model construction operation is simple, the formation and ablation repeatability of calcium oxalate crystals are good, the period is short, and the related indexes are convenient to quantify; the individual of the Chinese rice locust is more than 20 times larger than that of the fruit fly, the number of the Martes tube is large, the crystal is convenient to dissect, observe and separate, and the ablation process of the metabolic urinary calculus-calcium oxalate crystal is convenient to monitor in real time.

The invention regulates and controls the ablation speed of calcium oxalate crystals by controlling the dosage and the action time of the potassium citrate solution. Can be applied to the physiological function of potassium citrate in the urinary system and the change mechanism of the related anion/cation concentration in the ablation process of calcium oxalate to carry out systematic research. The invention constructs a good animal model for carrying out ablation and disease treatment of human metabolic urinary calculi, drug screening and pharmacokinetic research.

Drawings

FIG. 1 shows that calcium oxalate crystals in Ma-tube of Chinese rice locust are gradually ablated under the action of potassium citrate.

Detailed Description

Example 1:

1. collecting adult Chinese rice locust in a field rice field, putting the adult Chinese rice locust into a creel, adaptively feeding the adult Chinese rice locust in a climatic incubator for 2-3 days, and feeding the adult Chinese rice locust with fresh wheat seedlings in the period, wherein the preferable feeding conditions are as follows: the light and dark are 13:11 hours, the temperature corresponding to the photoperiod is 25 +/-2 ℃ and 21 +/-2 ℃, and the humidity is 70-80 percent;

2. the formula of the insect normal saline comprises: NaCl: 0.138M, Na₂HPO₄：0.0073 M, KH₂PO₄：0.00147 M，KCl：0.0027 M， pH=7.4；

3. Preparing 8.5 mM copper chloride solution and 10.0 mM potassium citrate solution with the insect normal saline, and sterilizing for later use by steam sterilization at 120 ℃ for 20 min;

4. rinsing body surface and microinjector with 75% alcohol for sterilization, picking adaptively raised Chinese rice locust, penetrating internode membrane from the 3 rd and 4 th abdominal nodes of Chinese rice locust with microinjector, and slowly injecting 8 μ L of copper chloride solution with concentration of 8.0 mM;

5. 1-2 days later, insects are dissected, and transparent calcium oxalate crystals with regular shapes are generated in Marsdenia tubes under a microscope;

6. preparing 12.8 mM potassium citrate solution with insect normal saline, and sterilizing with 120 deg.C steam for 20 min;

7. injecting 8 μ L potassium citrate solution with concentration of 7.0 mM into Chinese rice locust with calcium oxalate nodule generation, placing in a climatic incubator, and feeding with fresh wheat seedling;

8. after 12 hours of the Chinese rice locust injected with the potassium citrate solution, the decomposition of calcium oxalate crystals in the Martensis tube can be observed through a microscope; the disintegration or ablation process of calcium oxalate can be regulated by adjusting the dosage of potassium citrate and the feeding amount.

Example 2: in this example, the concentration of the cupric chloride solution injected into the abdominal cavity of a chinese rice locust was 7.0 mM, the injection amount was 4 μ L, the concentration of the potassium citrate solution injected after 1 day of the raising was 7.0 mM, the injection amount was 5 μ L, and the same effects as in example 1 were obtained except that the requirements were the same as in example 1.

Example 3: in this example, the concentration of the cupric chloride solution injected into the abdominal cavity of the locusta chinese was 6.0 mM, the injection amount was 7 μ L, the concentration of the potassium citrate solution injected after 1 day of the raising was 6.0 mM, the injection amount was 6 μ L, the other operational requirements were the same as those of example 1, and the calcium oxalate crystal ablation rate was slower and the period could be extended by 1 to 2 days as compared with example 1.

Example 4: in this example, the concentration of the cupric chloride solution injected into the abdominal cavity of the locusta chinese was 10.0 mM, the injection amount was 4 μ L, the concentration of the potassium citrate solution injected after 1 day of raising was 7.0 mM, the injection amount was 8 μ L, other operation requirements were the same as those of example 1, the calcium oxalate crystal ablation rate was accelerated, and the complete ablation was carried out 1 to 2 days earlier than that of example 1.

Claims

1. A construction method of an animal model for ablating metabolic urinary calculus comprises injecting potassium citrate solution with concentration of 6.7-12.8 mM and volume of 5-10 μ L into the animal model for constructing urinary calculus, and gradually disintegrating calcium oxalate crystals formed in a marquis tube of the animal model after injecting the potassium citrate solution for 12 hours until the calcium oxalate crystals completely disappear; the method is characterized by comprising the following steps of:

(1) adaptively feeding adult Chinese rice locust in a climatic incubator for 2-3 days by using fresh wheat seedlings; the breeding conditions are as follows: the period of light and dark is 13:11 hours, the temperature in the corresponding light is 25-28 +/-2 ℃, the temperature in the dark is 21-26 +/-2 ℃, and the humidity is 65-85%;

(2) spraying 75% alcohol on the body surface of Chinese rice locust for disinfection; sterilizing the syringe with 75% alcohol;

(3) injecting 4-8 μ L of copper chloride solution with concentration of 6.0-10.5 mM into internode membrane of 3 rd and 4 th abdominal nodes of Oxya chinensis (Oryza Glutinosa) by syringe;

(4) feeding Chinese rice locust injected with copper chloride solution in a climatic incubator for 1 day, injecting copper chloride solution 4-6 μ L, and feeding for 1-2 days; irregular calcium oxalate crystals are formed in the Mactra tube of the Chinese rice locust.

2. The method of claim 1, wherein the cupric chloride solution is prepared with normal saline insect solution at a concentration of 10.5 mM and steam sterilized at 120 deg.C for 20 min.

3. The method for constructing an animal model for ablating metabolic urinary calculi according to claim 2, wherein the formulation of the insect physiological saline is as follows: NaCl: 0.138M, KCl: 0.0027M, Na₂HPO₄：0.0073 M，KH₂PO₄：0.00147 M，pH=7.4。